PROJECT SUMMARY Protein lysine methylation is a reversible process controlled by lysine methyltransferases (KMTs or methyl writers) and lysine demethylases (KDMs or methyl erasers) in human cells. Most studies on these KMTs and KDMs have focused mainly on their functions inside the nucleus. More than half of these KMTs and KDMs, which are present in the nucleus are also localized in the cytoplasm but very little is known about their functions in this subcellular compartment. The abnormalities in these enzymes are directly associated with cancers, inflammation and other diseases. Despite the critical importance of the KDMs in different subcellular compartments, there is a substantial gap between their global analysis and effective methods available to achieve it. The long-term goal of this research program is to develop a mechanistic understanding of how ?lysine methylome? is maintained at a subcellular level and how these chemical markers modulate cell signaling. The current focus is to develop a new class of chemical tools that report on KDM activities in living cells at a subcellular level. This new family of chemical tools will be deployed to determine the activity of KDMs in different subcellular compartments, and their regulation by substrates, and local cofactors. The primary biological interests right now deal with roles of KDMs outside of the nucleus, in particular, the cytoplasm and mitochondria, while pursuing mechanistic studies in the context of cancer. The proposed research contains four innovations to decode the intracellular KDM activities with subcellular resolution. First, is the development of new small molecule probes with unique chemoselectivity towards lysine, and high stability towards hydrolysis, which is ideal for studying KDMs in cells. Second, is the development of molecular imaging agents to monitor the activity of KDMs and how they are regulated inside the cells. Third, is to determine the activity of the specific KDMs by incorporating recognition elements on the probe. Fourth, is the development of organelle selective molecular imaging agents to study the role of KDMs in different subcellular compartments and their subcellular localization, which is currently impossible to determine with existing techniques. These probes are capable of determining KDM activities and their role in various diseased states thus of immense therapeutic interest. This research will have extensive applications in biomedical field by providing a better understanding of KDMs functions in different subcellular compartments, the molecular mechanisms of diseases, thus assist in the discovery of novel protein biomarkers, and combination-therapy for the treatment of cancer.